Characterisation and expression of the Fasciolagigantica cathepsin L gene

International Journal for Parasitology 32 (2002) 1031–1042 www.parasitology-online.com

Characterisation and expression of the Fasciola gigantica cathepsin L gene q Hiroshi Yamasaki a,*, Reiko Mineki b, Kimie Murayama b, Akira Ito c, Takashi Aoki a a

b

Department of Parasitology, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo 113-0033, Japan Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo 113-0033, Japan c Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japan. Received 21 December 2001; received in revised form 26 February 2002; accepted 28 February 2002

Abstract The gene structure of a cathepsin L from Fasciola gigantica was characterised. The gene spans approximately 2.0 kb and comprises four exons and three introns and is a compact gene as in the cases of crustaceous and platyhelminth cathepsins L. Southern blot analysis suggested that a few copies of the genes are sparsely organised in the genome. Of the three intron insertion positions, two of which are in the same position as in the mammalian cathepsin L gene. Phylogenetic analysis revealed that F. gigantica cathepsin L forms a clade with those from Fasciola hepatica, but not with those from Spirometra erinacei and schistosomes. Putative TATA-boxes were found upstream of a transcription initiation site. The sequence analysis of the 5 0 -upstream of the transcript revealed that the cathepsin L gene is transcribed by cis-splicing fashion. Furthermore, the experiments using recombinant F. gigantica procathepsin L showed that it was processed to an enzymatically active cathepsin L by pH-dependent autocatalysis. However, the pro-peptide deleted cathepsin L showed no enzyme activity, indicating that the pro-region of F. gigantica procathepsin L is essential for the folding and/or refolding of functional cathepsin L. These results are consistent with the observations in mammalian cathepsin L and papain. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Fasciola gigantica; Cathepsin L; Gene structure; Gene family; Expression; In vitro processing

1. Introduction The liver flukes Fasciola hepatica and Fasciola gigantica are causative agents of fascioliasis in humans and ruminants, especially cattle, goat and sheep. The disease has been traditionally considered to be an important veterinary disease because of the substantial production and large economic losses in livestock production. In contrast, human fascioliasis has been linked to cases among livestock in the area concerned. However, it has been considered an increasingly important chronic disease since 1980 (Chen and Mott, 1990; Esteban et al., 1998; Mas-Coma et al., 1999). It has been reported that F. hepatica thiol-activated proteolytic enzymes secreted by migrating parasites cleave q The nucleotide sequence data reported in the present paper have been submitted to DDBJ, GenBank and EMBL databases with accession numbers AB010923 and AB010924. * Corresponding author. Present address: Department of Parasitology, Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asahikawa 078-8510, Japan. Tel.: 181-166-68-2421; Fax: 181-166-68-2429. E-mail address: [email protected] (H. Yamasaki).

host immunoglobulins (Chapman and Mitchell, 1982). Since then, the proteinases secreted by adult liver flukes have been characterised, but most studies agree that the major enzymes are cathepsin L-like cysteine proteases that are homologues of the mammalian lysosomal cathepsin L (Yamasaki et al., 1989; Rege et al., 1989; Dalton and Hefferman, 1989; Fagbemi and Hillyer, 1992; Yamasaki and Aoki, 1993; McGinty et al., 1993; Smith et al., 1993; Wijffels et al., 1994a; Dowd et al., 1994; Heussler and Dobbelaere, 1994; Hawthorne et al., 2000). It is considered that Fasciola cathepsin L may be involved in crucial biological functions such as host protein degradation, tissue penetration and immune evasion. For these reasons, the cathepsin L-like cysteine proteases of liver flukes have been potential targets as immunodiagnostic antigens for fascioliasis (Yamasaki et al., 1989; Fagbemi and Guobadia, 1995; O’Neill et al., 1999) or as vaccine candidates (Wijffels et al., 1994a; Dalton et al., 1996b). On the other hand, concerning genomic organisation of cathepsin L genes in invertebrates, studies on a kinetoplastid protozoon (Eakin et al., 1992), a malaria parasite (Rosenthal and Nelson, 1992), a fruit fly (Matsumoto et al., 1995), a

0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(02)00057-7

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larval cestode (Liu et al., 1996) and a shrimp (Boulay et al., 1998) have been reported. In the present paper, we describe the gene structure of a cathepsin L from F. gigantica and compare it with those of known cathepsins L from other organisms with a phylogenetic analysis. In addition, the function of the pro-region is also discussed using recombinant F. gigantica procathepsin L. 2. Materials and methods 2.1. Parasite Adult Fasciola worms were collected from a cattle naturally infected with the parasites at a local slaughterhouse in Tokyo. The parasites were washed several times in sterile PBS, pH 7.2, and immediately frozen in liquid nitrogen for RNA preparation or stored at 280 8C for DNA samples. The parasite used, F. gigantica, was identified based on the nucleotide sequences of cytochrome c oxidase subunit I and ribosomal RNA genes (Hashimoto et al., 1997). 2.2. Construction of the Fasciola genomic DNA library Genomic DNA was prepared from 1 g of a frozen worm, partially digested with Sau3AI, and fractionated into 5–20 kb fragments by sucrose density gradient method (Maniatis et al., 1982). The fragments were ligated to BamHI/EcoRI double-digested EMBL 3 lambda DNA, and allowed to form phage particles using an in vitro packaging kit (Stratagene). Approximately 6 £ 104 recombinant phages were screened using a digoxigenin-labelled Fasciola cathepsin L cDNA probe (Yamasaki and Aoki, 1993). 2.3. Southern blot analysis Genomic DNA was digested with different restriction enzymes whose cleavage sites are absent from the cathepsin L gene, separated on a 0.7% agarose gel, transferred to a positively charged nylon membrane (Boehringer Mannheim) and prehybridised at 68 8C for 2 h in hybridisation buffer containing 7% SDS. The filter was hybridised at 68 8C overnight with a digoxigenin-labelled probe. The 1.9-kb probe was prepared by PCR using primers based on the nucleotide sequences of F. gigantica cathepsin L gene (B22-2) shown in Fig. 2. Finally, the filter was washed at high stringency and exposed to X-ray film for 5–10 min. CSPD (Boehringer Mannheim) was used as a substrate for chemiluminescent detection.

the manufacturer’s instructions (Life Technologies Inc.) to determine the sequence of the 5 0 -untranslated region of the F. gigantica cathepsin L gene (Fg-CATL). Gene-specific antisense primers (GSP 1, 5 0 -TTGATGCCACAAATCATCATTCGAGCCAAG-3 0 and GSP 2, 5 0 -ATCTTCCCAAATATTTCGTCTGTG-3 0 ) were used for nested PCR. PCR was performed for 35 cycles of 1 min at 94 8C, 1 min at 55 8C, 2 min at 72 8C, and 4 min at 72 8C for extension. In order to clarify whether a spliced leader sequence is added at the 5 0 end of the transcript of Fg-CATL, mRNA was reversibly transcribed with M-MLV reverse transcriptase (Gibco BRL) and oligo(dT) primer (Boehringer Mannheim), followed by PCR performed at 94 8C for 1 min, 55 8C for 1 min, 72 8C for 2 min, for 35 cycles using a Fasciola spliced leader primer (5 0 -AACCTTAACGGTTCTCTG-3 0 , Davis et al., 1994) and GSP 1 primer. mRNA was electrophoresed in a 1% agarose gel, blotted onto a positively charged nylon membrane, and then hybridised with a digoxigenin-labelled cathepsin L (B22-2) cDNA probe overnight at 42 8C. The probe was prepared by PCR using primers based on the nucleotide sequences of F. gigantica cathepsin L gene (B22-2) and cDNA prepared using cDNA synthesis kit (Pharmacia). The hybridisation buffer used was 50 mM phosphate buffer, pH 7.0, containing 5£ standard saline citrate (SSC), 7% SDS, 0.1% sarcosine, 50% formamide and yeast total RNA (50 mg/ml). The filter was washed extensively with 2£ SSC/0.1% SDS, 0:1£ SSC/ 0.1% SDS at 42 8C, and chemiluminescent detection was carried out using CSPD as a substrate.

2.5. DNA sequencing A DNA fragment derived from a positive clone (B22-2 clone) was digested with restriction enzymes as indicated in Fig. 1, and the resulting fragments were subcloned into pUC18. The nucleotide sequences of the genomic DNA were determined by the dideoxynucleotide chain termination method using Dye Primer and Dye Terminator Cycle Sequencing kits (Applied Biosystems Inc.) and an ABI DNA sequencer 373A. The nucleotide sequence of the PCR-amplified cathepsin L gene (B7-3) was also determined by same protocol. Sequence data were analysed using Genetix-Mac, and EMBL/GenBank database searches were performed with the FASTA program.

2.6. Sequence alignment and phylogenetic analysis

0

2.4. RNA preparation, 5 -RACE and Northern blot analysis Total RNA was extracted from a frozen worm (0.8 g weight) by the acid guanidinium thiocyanate phenol-chloroform method (Chomczynski and Sacchi, 1987). Poly(A) 1 RNA was purified on an oligo(dT) cellulose gel column (Pharmacia Amersham Biotech, USA). 5 0 -rapid amplification of cDNA ends (5 0 -RACE) was performed according to

The alignment of sequences was carried out using CLUSTAL W program available over the World Wide Web (http://www.ddbj.nig.ac.jp/E-mail/homology.html). Phylogenetic tree was inferred with the neighbour-joining method (Saitou and Nei, 1987) using TreeView PPC software (version 1.5.3). Confidence values for each branch were determined by 1000 bootstrap replications.

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Fig. 1. Genomic organisation of Fasciola gigantica cathepsin L gene and sequencing strategy. In the cDNA, dotted, open and striped boxes indicate regions encoding the signal sequence, pro-peptide and mature enzyme domains, respectively. In the genomic clone, closed boxes indicate exons and solid bars denote introns. Bm, BamHI; M, MvaI; C, ClaI; St, StyI; Sp, SspI; A, AgeI; E, EcoO109I; B, BsmI. Arrows indicate the direction of sequencing.

2.7. Expression and refolding of the recombinant procathepsin L and pro-peptide deleted cathepsin L Two cDNAs encoding F. gigantica procathepsin L and pro-peptide deleted cathepsin L (B22-2) were amplified by PCR using cDNA as described above. Primer sets used were pro 5 0 -primer/3 0 -primer, and mCL 5 0 -primer/3 0 -primer based on the nucleotide sequence of Fg CATL (B22-2, AB010923). PCR was performed using AmpliTaq DNA polymerase (Perkin Elmer) as follows: 95 8C, 1 min; 55 8C, 1 min; 72 8C, 2 min; 35 cycles. The PCR product was subcloned into pT7Blue T-vector (Novagene, USA) and sequenced. cDNA corresponding to B22-2 clone was inserted into the pGEX-4T-1 expression vector (Pharmacia Amersham Biotech), and designated pGEX/proCATL for procathepsin L or pGEX/mCATL for pro-peptide deleted cathepsin L. Each construct was then introduced into BL21 Escherichia coli strain (Novagene). The expression of glutathione S-transferase (GST) fused with either procathepsin L or pro-peptide deleted cathepsin L was induced by adding a final concentration of 1 mM isopropyl-b-d(2)thiogalactopyranoside (IPTG) for 2–3 h at 37 8C. Cells from 1 l culture were harvested, and the cell pellet was resuspended in chilled PBS containing 0.1% Triton X100 and 1% sarcosine, and then sonicated using a sonifier (Branson). The resulting suspensions were centrifuged at 15,000 rpm for 10 min, and the pellets were rinsed in several times in PBS and resuspended in 20 ml of 50 mM Tris–HCl, pH 8.0, containing 50 mM NaCl, 5 mM EDTA and 10 mM

dithiothreitol (DTT). Urea was added to a final concentration of 8 M to solubilise the suspension completely. Refolding was performed according to a previously described procedure (Smith and Gottesman, 1989). Briefly, the ureasolubilised GST-fusion proteins were slowly added dropwise (2 ml/h) into 400 volumes of 50 mM potassium phosphate buffer, pH 10.7, containing 5 mM EDTA, 0.1 mM oxidised glutathione and 1 mM reduced glutathione to a final protein concentration of approximately 10 mg/ml, and stirred at 4 8C overnight. The solutions of solubilised proteins were then adjusted to pH 8.0 and concentrated to 1/ 300 of the original volume using an Amicon cell ultrafiltration unit. After concentration, the solutions were centrifuged briefly for further experiments. 2.8. Purification of recombinant and native cathepsins L The refolded GST-fusion proteins were cleaved with thrombin to remove GST according to the manufacturer’s manual and the recombinant procathepsin L was dialysed against 10 mM Na2HPO4/1.8 mM KH2PO4, pH 8.0, containing 140 mM NaCl, 2.7 mM KCl and 4 M urea, and the resulting solution were applied to a Sephacryl S-200 HR gel chromatography column (1:5 £ 120 cm) equilibrated with same buffer. The eluates were analysed by SDS– PAGE (Laemmli, 1970). In order to compare the activities of recombinant and native cathepsin L, native cathepsin L was partially purified from F. gigantica adult worms. Briefly, 1.5 g of lyophilised parasites were homogenised

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in 40 mM sodium acetate buffer, pH 3.9, containing 150 mM NaCl, 1 mM EDTA, and 1 mM DTT. The resulting supernatant was dialysed against the same buffer and applied to TSK-Gel 3000SW column (21:5 £ 600 mm) in a high-performance liquid chromatograph (Shimadzu, Model LC-6A). Fractions showing activities toward carbobenzoxyl-l-phenylalanyl-l-arginine 4-methyl-coumaryl amide (Z-Phe-Arg-MCA, Peptide Institute) were pooled for further experiments. 2.9. Enzyme assay Cathepsin L activity was assayed according to the method of Barrett and Kirschke, (1981). Briefly, 10 ml of recombinant or native cathepsin L in 80 ml of 80 mM sodium acetate buffer, pH 5.5, containing 8 mM l-cysteine and 4 mM EDTA were preincubated at 37 8C for 5 min, and the enzyme reaction was initiated by adding 10 ml of 1 mM Z-Phe-Arg-MCA at 37 8C for 10 min, and terminated by adding 100 ml of 5% SDS. The 7-amino-4-methyl-coumarin released was measured using a fluorescence spectrophotometry (Hitachi, Model 850) at an excitation wavelength of 370 nm and emission wavelength of 460 nm. 2.10. NH2-terminal sequencing of recombinant procathepsin L, pro-peptide deleted- and processed cathepsins L Both GST-fused procathepsin L and cathepsin L were cleaved with thrombin, electrophoresed, and then were blotted onto a PVDF membrane for NH2-terminal sequencing according to the method of Matsudaira (1987). The NH2-terminal amino acid sequence of recombinant cathepsin L processed in vitro was also electrophoresed. The target bands were cut and then applied to a Protein Sequencer (Applied Biosystems, Model 477A). 2.11. Protein assay The protein content was determined by the Bradford method using a protein assay kit (BioRad). 3. Results 3.1. Structure of the F. gigantica cathepsin L gene Recombinant clones (6 £ 104 ) were screened with a digoxigenin-labelled Fasciola cathepsin L cDNA probe (Yamasaki and Aoki, 1993), and a positive clone (B22-2) with a 16 kb DNA fragment was isolated. The DNA fragment was digested with BamHI, a 4.3 kb fragment hybridised with the probe was obtained, and 3,766 bp including a 5 0 -flanking region of the fragment were determined. Fig. 1 illustrates the schematic structure of the F. gigantica cathepsin L gene (Fg-CATL) and sequence strategy. The nucleotide and deduced amino acid sequences of the Fg-CATL (B22-2, AB010923) are shown in Fig. 2. The gene consists

of four exons and three introns spanning approximately 2.0 kb in the genome. The length of each exon varies from 378, 222, 158, and 217 bp in exons 1, 2, 3 and 4, respectively. Exon 1 encodes 15 amino acid residues of the signal sequence, 90 amino acid residues of the pro-region, and the first 21 residues of the mature enzyme. The remainder of the mature enzyme is encoded by exons 2–4. Cys 27, His 164 and Asn 184, which form the catalytic triad in the active sites, are encoded in exons 2 and 4, respectively. The intron breakpoints are not found at the junction of the pre-, propeptide and mature enzyme domains, indicating that the gene structure does not correspond to the functional units of the protease as well other cathepsin L (Ishidoh et al., 1989). The length of each intron varies from 53, 163 and 694 bp in introns 1, 2 and 3, respectively. Furthermore, introns 1 and 2 interrupt the open reading frame between two codons (type 0), whereas intron 3 interrupts the coding sequence after the second nucleotide of a codon (type 2) (Patthy, 1987). The exon–intron boundary sites, determined by comparison with the cDNA sequence, are all consistent with the GT-AG rule (Breathnach and Chambon, 1981). In the 5 0 -flanking region of Fg-CATL, two putative TATA boxes were found 58 and 68 nucleotides upstream of a transcription initiation site (Fig. 2), one of which might be involved in the transcription activity of the gene. However, no CCAAT (CAT box) that enhances the transcription activity was found in this region, as in the case of Spirometra erinacei cathepsin L gene (Liu et al., 1996). Southern blot analysis of F. gigantica genomic DNA revealed two or three signals in each of the enzymes used (Fig. 3). Apart from the genomic clone (B22-2), another1.8kb Fg-CATL was co-amplified (data not shown) when FgCATL (B22-2) was amplified by PCR using genomic DNA for probe preparation. Sequence analysis revealed the gene, termed Fg-CATL (B7-3, accession number AB010924), is distinct from Fg-CATL (B22-2) at the nucleotide and amino acid levels. The remarked difference was observed in length and sequence in the third intron, the sizes of 582 and 694 bp were in the B22-2 and B7-3 Fg-CATLs, respectively. In Northern blot analysis, an approximately 1.0 kb transcript was detected when a B22-2 cathepsin L cDNA was used as a probe (Fig. 4). Sequence analysis of the 5 0 -untranslated region of the Fg-CATL mRNAs revealed a predicted transcription initiation site at an adenine 14 nucleotides upstream of the translation initiation site. 3.2. Amino acid sequence characterisation of F. gigantica cathepsin L and homology with other known cathepsin L Fig. 5 shows the aligned sequences and intron insertion positions of cathepsins L from F. gigantica, S. erinacei plerocercoid (Liu et al., 1996), shrimp (Boulay et al., 1998) and rat (Ishidoh et al., 1989). The amino acid sequences of F. gigantica cathepsins L (Fg CATL B22-2 and B7-3) are homologous to those of cathepsins L from other eukaryotes. The regions containing the Cys 27, His 164

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and Asn 184 residues at the active sites are highly conserved. Since it is confirmed that a cathepsin L (B22-2) is expressed in the F. gigantica adult worms, the cathepsin L described here is Fg CATL (B22-2). The ERFNIN motif (Karrer et al.,

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1993) was found, but the first amino acid of the motif, Glu, was replaced by Val (grey box in Fig. 2 and overlined a in Fig. 5). Most recently, an evolutionarily conserved tripartite tryptophan motif has been identified in cathepsin L-like

Fig. 2. Nucleotide and deduced amino acid sequences of Fasciola gigantica cathepsin L (B22-2). Nucleotides are numbered from the transcription initiation site (11). The amino acid sequence deduced from the nucleotide sequence is shown below the nucleotide sequences and is numbered from the first amino acid (Arg 11) of the mature enzyme. Intron sequences are shown in lowercase letters. Putative TATA boxes and a polyadenylation addition signal are in grey boxes. The asterisk indicates a translation termination codon. Arrows a and b indicate putative cleavage sites for signal sequence and the pro-peptide. Active site residues (Cys 27, His 164 and Asn 184) are in black boxes. The ERFNIN motif (Val 270 to Asn 251) and the putative intramolecular processing motif (Gly 238 to Asp 232) are in grey and black boxes, respectively. The three aromatic residues (Trp 285, Trp 282 and Trp 262) in a tripartite tryptophan motif are underlined. The NH2-terminal sequence of the recombinant cathepsin L processed in vitro is underlined (Glu 24 to Gln 21). GSP 1 and 2 primers were used for 5 0 -rapid amplification of cDNA ends. The underlined region (nucleotides 60–83), mCL 5 0 - and 3 0 -primers were used to amplify the procathepsin L and the pro-peptide deleted cathepsin L cDNAs, respectively.

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Fig. 3. Southern blot analysis of Fasciola gigantica cathepsin L gene. Fasciola gigantica genomic DNA (1.2 mg) was digested completely with either BamHI, HindIII, NcoI or XhoI, electrophoresed, transferred, and then hybridised with a digoxigenin-labelled Fasciola preprocathepsin L genomic clone as a probe. Digoxigenin-labelled DNA size markers are indicated on the right.

cysteine proteases (Kreusch et al., 2000). In F. gigantica cathepsin L, this motif is completely conserved (doubleunderlined in Fig. 2 and closed triangles in Fig.5). Another sequence motif, GNFD, which may be involved in intramolecular processing in propapain (Vernet et al., 1995), is also highly conserved although Phe 238 is replaced by Leu 234 in F. gigantica cathepsin L (black box in Fig.2 and overline b in Fig. 5). As shown in Fig.2, Fg-CATL has three introns, two of which is located in the same position as in rat, and one of which is same as in rat and shrimp cathepsins L (Fig. 5). Interestingly, there is no common intron insertion posi-

Fig. 4. Northern blot analysis of a Fasciola gigantica cathepsin L transcript. mRNA (0.6 mg) was electrophoresed in a 1% agarose gel containing 50% formamide, transferred to a nylon membrane, hybridised with a digoxigenin-labelled Fasciola cathepsin L cDNA (B22-2) as a probe, and exposed to X-ray film for 5 min. Digoxigenin-labelled RNA size markers are indicated on the right.

tion between Fg-CATL and S. erinacei plerocercoid cathepsin L gene though these parasites belong to taxonomically the phylum platyhelminths. The region around the catalytic site Cys 27 is generally split by an intron. In F. gigantica cathepsin L, the junction is located close to the Cys 27 residue, as in rat cathepsin L, whereas the sites in S. erinacei and shrimp cathepsins L are located before and after the Cys 27 residue, respectively. Table 1 shows the amino acid homologies of Fg CATL (B22-2) with cathepsins L from parasitic platyhelminths, a shrimp and a mammal. Fg CATL (B22-2) is highly homologous with F. gigantica cathepsin L (Fg CATL-A, Grams et al., 2001) and F. hepatica cathepsin L1 (Fh CATL-1, Roche et al., 1997) with homologies of 94.8 and 91.1%, respectively, not but with F. gigantica cathepsin L (Fg CATL-D, Grams et al., 2001) and F. hepatica cathepsin L2 (Fh CATL-2, Dowd et al., 1997). Fg CATL (B22-2) shows lower homologies (42.1–44.8%) with those of a larval cestode, schistosomes, shrimp and rat. 3.3. Phylogenetic analysis of cathepsin L from F. gigantica A phylogenetic analysis of 25 members of the papain superfamily revealed that the cathepsins L from F. gigantica form a monophyletic cluster with those of F. hepatica (Fig. 6). The two subgroups, representing by Fg CATL-D and Fh CATL-2, and by Fg CATL (B22-2) and Fh CATL-1, are seemed to be similar divergence between each other that do the cathepsins L of schistosomes and F. hepatica. Interestingly, cathepsins L from the liver flukes form a separate clade that contains the cathepsins L from Schistosoma japonicum (Sj CATL-2, Day et al., 1995), Schistosoma mansoni (Sm CATL-2, Dalton et al., 1996a) and S. erinacei that belong to the same phylum as well as the liver flukes. 3.4. Expression and in vitro processing of the recombinant procathepsin L Fig. 7 shows SDS–PAGE analysis of proteins produced in non-induced and induced cells carrying either pGEX/ proCATL or pGEX/mCATL. GST-procathepsin L and GST-cathepsin L were expressed as insoluble proteins with molecular masses of 64 and 52 kDa, respectively. The fusion proteins were solubilised in 8 M urea and renatured by the procedure described in Section 2. The refolded GST-fusion proteins were then treated with thrombin to separate target proteins from GST. Finally, 38 and 26 kDa proteins were separated on SDS–PAGE, and the sequences of the NH2-terminal first 10 amino acids of each protein coincided completely with those deduced from the procathepsin L and mature form cathepsin L cDNAs. Once F. gigantica procathepsin L was refolded, it showed increasing enzyme activities against a fluorogenic substrate, Z-Phe-Arg-MCA, over time. As shown in Fig. 8, the hydrolysing activities increased remarkably at pH 4.5–5.5. However, the enzyme activities were lower at other pH and no activity was detected at pH 8.0. The processing occurs by a

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Fig. 5. Alignment of the predicted amino acid sequences and intron insertion positions of cathepsins L from Fasciola gigantica, Spirometra erinacei, shrimp and rat. Cathepsin L genes whose intron insertion positions are determined are selected. The catalytic triad residues are marked with asterisks, and gaps (-) are introduced to maximise alignment. Arrows a and b indicate the putative cleavage sites of the signal sequence and pro-peptide of F. gigantica cathepsin L, respectively. Intron insertion positions are indicated by closed circles. The ERFNIN and the intramolecular processing motifs are shown by overlines a and b, respectively. The tripartite tryptophan residues are denoted by closed triangles. Identical amino acid residues are shown in black boxes.

pH-dependent autocatalytic mechanism in vitro and it seems to occur in multiple steps based on the results by Western blotting (data not shown). Interestingly, the processed active cathepsin L degraded GST in the reaction mixture after overnight incubation at pH 5.5. In addition, the processing reaction was completely inhibited in the presence of an irreversible inhibitor, E-64 (Fig. 8), indicating that the interaction with the prodomain of procathepsin L with the inhibitor results in the inhibition of autocatalysis of the enzyme. The specific activity of the processed cathepsin L was 4,282 nmol/mg protein per minute, corresponding to 80% of that of native cathepsin L (5,308 nmol/mg protein per minute). Km values for recombinant and native cathepsin L toward Z-Phe-Arg-MCA were 83.3 and 23.3 mM, respectively. The processed cathepsin L did not show any activities against Z-Arg-Arg-MCA, a substrate for cathepsin B, and Arg-MCA for cathepsin H as well as native F. gigantica cathepsin L (data not shown). Furthermore, the sequencing of the 26 kDa-processed cathepsin L revealed the NH2-terminal to be Glu 24-Ala 23-Asn 22-Asn 21-Arg 11-Val 12-Pro 13Asp 14 (underlined in Fig. 2), corresponding to a fouramino-acid extension at the NH2-terminal of native F. gigantica cathepsin L.

4. Discussion We describe here the genomic structure and sequence analysis of F. gigantica cathepsin L and the in vitro processing of the recombinant F. gigantica procathepsin L. Sequence analysis revealed that Fg-CATL is as a basically similar structure to the other cathepsin L genes. However, Fg-CATL contains fewer introns compared with those of its mammalian counterparts and is a compact gene as in the case of cathepsin L from a platyhelminth S. erinacei (Liu et al., 1996). Previously sequenced cathepsin L genes from protozoan parasites are entirely devoid of any introns (Rosenthal and Nelson, 1992; Eakin et al., 1992). Regarding intron insertion positions, a junction close to the catalytic residue (Asn 185) found in rat cathepsin L is absent in F. gigantica cathepsin L. The junction between Val 200 and Glu 201 is conserved in shrimp and rat cathepsins L and in rat cathepsin H. It is interesting to note that the intron insertion positions are similar to those in mammalian and crustaceous cathepsins L rather than to those in the taxonomically closely related S. erinacei. Southern blot analysis revealed that the Fg-CATL gene family comprises only a few copies. It was difficult to esti-

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Fg CATL (B22-2) Fg CATL (B7-3) a Fg CATL-A b Fg CATL-D b Fh CATL-1 c Fh CATL-2 d Se CATL e Sj CATL-2 f Sm CATL-2 g Shrimp CATL h Rat CATL i a b c d e f g h i

a

Fg CATL (B22-2)

Fg CATL (B7-3)

Fg CATL-A

Fg CATL-D

Fh CATL-1

Fh CATL-2

Se CATL

Sj CATL-2

Sm CATL-2

Shrimp CATL

100.0 78.8 94.8 77.3 91.1 77.0 43.6 43.9 44.8 44.5 42.1

100.0 81.3 77.9 80.1 77.9 43.0 43.6 45.4 41.3 40.5

100.0 78.5 93.6 77.3 45.0 44.6 46.1 45.4 43.0

100.0 77.3 92.3 45.3 44.6 44.1 43.6 39.4

100.0 77.3 45.0 44.9 46.7 45.1 43.4

100.0 46.6 43.4 45.4 43.9 41.9

100.0 43.6 46.1 46.2 46.0

100.0 75.7 40.4 40.1

100.0 44.6 44.7

100.0 51.5

Fasciola gigantica cathepsin L descibed in the present study. Fasciola gigantica cathepsin L (Grams et al., 2001). Fasciola hepatica cathepsin L (Roche et al., 1997). Fasciola hepatica cathepsin L (Dowd et al., 1997). Spirometra erinacei cathepsin L (Liu et al., 1996). Schistosoma japonicum cathepsin L (Day et al., 1995). Schistosoma mansoni cathepsin L (Dalton et al., 1996a). Penaeus vannamei cathepsin L (Boulay et al., 1998). Rattus norvegicus cathepsin L (Ishidoh et al., 1989).

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Table 1 Amino acid homologies among selected cathepsins L

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Fig. 6. Phylogenetic tree of cathepsins L from Fasciola gigantica, Fasciola hepatica and other organisms. Unrooted neighbour-joining tree was prepared using the amino acid sequences of representative members of the papain superfamily. Numbers adjacent to branches represent bootstrap values. The bar indicates the numbers of substitutions per site. The cathepsins L used were from F. gigantica (Fg CATL-A, AF112566, Fg CATL B-D, AF239264-6), F. hepatica (Fh CATL-1, U62288; Fh CATL-2, U62289), Penaeus vannamei (Shrimp CATL, Y14965), Rat (Rat CATL, Y00697), human (Human CATL, X12451), Spirometra erinacei (Se CATL, D63670), Schistosoma mansoni (Sm CATL-1, U07345; Sm CATL-2, Z32529), Schistosoma japonicum (Sj CATL-1, U38475; Sj CATL-2, U38476), Plasmodium falciparum (Pf FALPN, M81341), Leishmania mexicana (Lm CATL, Z14061), Trypanosoma cruzi (Tc CZPN, X54414) and papain (M15203).

mate exact copy numbers, but at least three copies exist. Grams et al. (2001) reported that a cathepsin L gene family comprising an estimated 10 related cathepsin L genes exists in F. gigantica. From these observations, it is obvious that cathepsin L genes form a gene family in the F. gigantica as in the cases of F. hepatica (Dowd et al., 1994; Heussler and Dobbelaere, 1994). This is similar to the degree of diversity reported for cathepsin L genes in S. mansoni (Dalton et al., 1996a) and a coleopteran insect, Sitophilus zeamais (Matsumoto et al., 1997). However, it is not clear why the closely related cathepsin L genes have been evolved in these liver flukes. Possibly, gene duplication of cathepsin L gene occurred in the common ancestry for both F. gigantica and F. hepatica. After species differentiation, cathepsin L genes might have been diversified by gene conversion in each Fasciola species. The existence of multiple cathepsin L genes may suggest that the cathepsin L family has diverse biological functions at different developmental stages or tissues although it is unclear whether multiple transcripts

are translated into different cathepsins L. A cathepsin L derived from the B22-2 clone is expressed as a predominant enzyme in F. gigantica adult worms (data not shown) and can degrade host haemoglobin as a physiological substrate as in the case of Fasciola sp. (Yamasaki et al., 1989, 1992). However, the enzyme cannot be cleaved intact host erythrocyte membrane (unpublished data). As far as we examined, there is no direct evidence that another cathepsin L (B7-3) is expressed in the F. gigantica adult worms. It is also known that a 37-nucleotide spliced leader sequence is added at the 5 0 -ends of F. hepatica mRNAs, indicating that trans-splicing is likely to be a common feature in trematodes and perhaps other flatworms (Davis et al., 1994). However, 5 0 -RACE or PCR results using a F. hepatica spliced leader primer did not support the feature mentioned above, indicating that Fg-CATL is transcribed by a cis-splicing mechanism. In the platyhelminth, it is interesting whether cysteine protease genes including cathepsin L are transcribed by cis-splicing fashion or not.

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Fig. 7. SDS–PAGE analysis of recombinant procathepsin L and cathepsin L fused with glutathione S-transferase (GST) expressed in Escherichia coli. Proteins from non-induced and induced cells were subjected to SDS–PAGE (10–20% gradient gel) to monitor the expression of GST-fusion proteins. Arrows indicate GST fused with procathepsin L (64 kDa) and cathepsin L (52 kDa), respectively. Sup and Ppt are soluble and insoluble fractions, respectively. Size markers are indicated on the left.

In the pro-peptide regions of cysteine proteases belonging to a cathepsin L subfamily, a consensus sequence exists comprising conserved amino acids interspersed with variable ones, EX3RX2(V/I)FX2NX3IX3N (Karrer et al., 1993). The ERFNIN motif, which is considered to be a functional unit involved in the inhibition of enzyme activity, was found in F. gigantica cathepsin L, although the first amino acid of the motif, Glu, is replaced by Val. An evolutionarily conserved tripartite tryptophan motif has been identified in cathepsin L-like cysteine proteases (Kreusch et al., 2000). The motif was shown to be of importance for the maintenance of the prodomain structure of procathepsin S. X-ray crystallographic studies of human procathepsin L showed that cathepsin L-like cysteine proteases contain two a helices crossing each other and the three aromatic residues contribute to a small core surrounded by hydrophobic residues at the point of intersection (Coulombe et al., 1996). The tripartite tryptophan motif, Trp 285, Trp 282 and Trp 262, is completely conserved in F. gigantica cathepsin L, suggesting that the motif may play important roles in the maintenance of the prodomain structure. The experiments using a recombinant procathepsin L revealed that the in vitro activation of procathepsin L occurs by pH-dependent autocatalysis. Furthermore, the NH2-terminal sequencing revealed that the 26 kDa-processed cathepsin L has Glu 24-Ala 23-Asn 22-Asn 21-Arg 11-Val 12-Pro 13Asp 14, corresponding to a four amino acid extension at the NH2-terminal of native F. gigantica cathepsin L. The difference could be due to differences in the pH-dependent autocatalytic processing in vitro and the cleavage site specificity

of the enzyme(s) involved in intracellular processing in vivo. In contrast to procathepsin L, the pro-peptide deleted recombinant cathepsin L showed no enzyme activity against any fluorogenic substrates. The GNFD motif in the prodomain of the propapain could participate in pH-dependent intramolecular processing (Vernet et al., 1995). A similar motif is found in F. gigantica procathepsin L, although Phe 238 in propapain is replaced by Leu 234 in F. gigantica cathepsin L. Considering the existence of a potential intramolecular processing motif in the pro-peptide region, it may be reasonable that pro-peptide deleted cathepsin L did not show any enzyme activity. A part of the pro-peptide is also essential for the proper folding of functional cathepsin L in F. gigantica. The pro-peptide also plays essential roles for protein folding, endoplasmic reticulum exit, stability, and mannose phosphorylation in mouse procathepsin L (Tao et al., 1994). As the function of the pro-peptide, Roche et al. (1999) reported that the pro-peptide of the F. hepatica cathepsin L (Fh CATL-1) showed a very potent and specific inhibition against the Fh CATL-1. This is considered that a part of the pro-peptide enters the substrate binding cleft in a reverse orientation to natural substrate and blocks access to the active site (Coulombe et al., 1996). In contrast to mammalian cathepsin L, it is very interesting that F. gigantica cathepsin L lacks N-linked sugar moiety. It has been reported that a cathepsin L from Japa-

Fig. 8. In vitro processing of procathepsin L. The purified recombinant procathepsin L was preincubated at 37 8C in buffers of varying pH for the times indicated, and the hydrolysing activities against Z-Phe-ArgMCA were assayed and plotted versus the corresponding preincubation time. pH 3.0 (- -W- -), pH 3.5 (- -O- -), pH 4.0 (—A—), pH 4.5 (—O—), pH 5.0 (—B—), pH 5.5 (—X—), pH 6.0 (—K—), pH 6.5 (- -B- -), pH 8.0 (- -X- -), pH 5.0 in the presence of E-64 (- -K- -). Each bar represents the average ^ SD for two independent experiments.

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nese Fasciola sp. localises in the secretory granules in intestinal epithelial cells of the worms (Yamasaki et al., 1992; Yamasaki and Aoki, 1993). This suggests that the intracellular transport mechanism of F. gigantica cathepsin L is different from those of mammalian cathepsins L. In order to clarify the function of the pro-peptide in the intracellular transport mechanism of F. gigantica cathepsin L, experiments using deletion mutants of F. gigantica procathepsin L fused with a green fluorescent protein are currently in progress. Acknowledgements We thank Professor Dr Takeshi Agatsuma for the molecular identification of the Fasciola parasite, and Naomi Mori, Koichi Kawakami and Mami Kaneko for technical assistance. This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan (Nos. 05670241,07670291, 09670271). References Barrett, J.A., Kirschke, H., 1981. cathepsin B, cathepsin H and Cathepsin L. Methods Enzymol. 80, 535–61. Boulay, C.L., Sellos, D., Wormhoudt, A.V., 1998. Cathepsin L gene organization in crustaceans. Gene 218, 77–84. Breathnach, R., Chambon, P., 1981. Organization and expression of eukaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349–83. Chapman, C.B., Mitchell, G.F., 1982. Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Vet. Parasitol. 11, 165–78. Chen, M.G., Mott, K.E., 1990. Progress in morbidity due to Fasciola hepatica infection. Trop. Dis. Bull. 87, 1–37. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–9. Coulombe, R., Grochulski, R., Sivaraman, J., Menard, R., Mort, J.S., Cygler, M., 1996. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. EMBO J. 15, 5492–503. Dalton, J.P., Hefferman, M., 1989. Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161–6. Dalton, J.P., Clough, K.A., Jones, M.K., Brindley, P.J., 1996a. Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni. Infect. Immun. 64, 1328–34. Dalton, J.P., McGonigle, S., Rolph, T.P., Andrews, S.J., 1996b. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinase and hemoglobin. Infect. Immun. 64, 5066–74. Davis, E.R., Singh, H., Botka, C., Hardwick, C., el Meanawy, M.A., Villanueva, J., 1994. RNA trans-splicing in Fasciola hepatica. Identification of a spliced leader (SL) RNA and SL sequences on mRNAs. J. Biol. Chem. 269, 20026–200230. Day, S.R., Dalton, J.P., Clough, K.A., Leonardo, L., Tiu, W.U., Brindley, P.J., 1995. Characterization and cloning of the cathepsin L proteinases of Schistosoma japonicum. Biochem. Biophys. Res. Comm. 217, 1–9. Dowd, A.J., Smith, M.A., McGonigle, S., Dalton, J.P., 1994. Purification and characterization of a second cathepsin L proteinase secreted by the trematode Fasciola hepatica. Eur. J. Biochem. 223, 91–98. Dowd, A.J., Tort, J., Roche, L., Ryan, T., Dalton, J.P., 1997. Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expres-

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